The JAK-STAT pathway is an essential signaling mechanism that initiates immune responses against pathogen infections. The intrinsic homeostatic regulation of JAK-STAT signaling is critical for maintaining immune homeostasis. Previous studies have shown that aberrant activation of the invertebrate JAK-STAT pathway leads to inflammation-like symptoms. Understanding the homeostatic mechanisms of this pathway in invertebrates is of significant interest. Pacific white shrimp (Penaeus vannamei) is one of the most extensively studied invertebrates in immune system research. In this study, we demonstrate that the shrimp microRNA-34 (miR-34) attenuates JAK-STAT pathway activation by targeting JAK, thereby inhibiting STAT phosphorylation and nuclear translocation. Interestingly, miR-34 expression is directly regulated by STAT, forming a negative feedback regulatory loop in the JAK-STAT pathway. Disrupting this loop results in excessive JAK-STAT pathway activation and immune overactivation, exacerbating inflammation caused by Vibrio parahaemolyticus infection in shrimp. This study provides new insights into the regulatory mechanism of the JAK-STAT pathway and its roles in maintaining immune homeostasis in invertebrates.